The focus of this research is the small nucleolar RNA protein machines called snoRNPs. Most snoRNPs produce modified nucleotides (of unknown function) in rRNA. A few others facilitate cleavage of pre- rRNA, probably by mediating folding of pre-rRNA. Two types of modifying snoRNPs exist, which form 2'-O-methylated nucleotides (Nm) and pseudouridines (psi) at specific sites. Specificity is provided by snoRNA guide sequences. We have determined that rRNA function can be impaired by blocking psi formation, and remarkably, by introducing Nm modifications at new sites. The specific aims of the research are: 1) to determine the role of snoRNA-directed nucleotide modification in ribosomal RNA; 2) to define the molecular functions of catalytically active snoRNA-associated proteins, and; 3) to develop snoRNA-directed modification as a strategy for probing RNA structure and function in vivo. The role of nucleotide modification will be determined by systematically blocking modifications in selected domains, and then evaluating rRNA maturation, rRNP assembly and ribosome function. The catalytic proteins to be studied include-- the two modifying enzymes, an RNA helicase required for rRNA processing, and two nucleoplasmic helicases required for snoRNP biogenesis. Genetic and biochemical strategies will be used to identify precise functions where unknown, and to define the mechanisms of action. To further assess the potential for using snoRNP- directed modification for mapping RNA function, we will extend an rRNA probing study in progress by targeting libraries of guide snoRNPs to new domains in rRNA. Several small RNAs thought to pass through the nucleolus will also be targeted, to determine the range of modifiable substrates. Finally, we will determine if snoRNP-directed modification can block growth of pathogenic yeast and disrupt HIV replication.